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. 2020 Nov 9;94(23):e01229-20. doi: 10.1128/JVI.01229-20

FIG 5.

FIG 5

The IRE1α-XBP1 signaling pathway upregulates the expression of SCD1 and promotes viral replication. (A and B) A549 cells were infected with lentivirus containing scramble shRNA (shScr), shXBP1-1, or shXBP1-2 and selected by puromycin for 1 week. Then, the cells were infected with ZIKV at an MOI of 3 and collected at 24 h p.i. to measure the expression of XBP1 (A) and the splicing of XBP1 (B). (C to E) ZIKV replication levels in XBP1 knockdown cells. shScr-, shXBP1-1-, and shXBP1-2-expressing cells were infected with ZIKV at an MOI of 3. Then, the cells and supernatants were harvested at 24 h p.i. to detect viral RNA levels (C), viral E protein levels (D), and viral yields (E) as described above. (F) SCD1 expression levels in IRE1αKo and IRE1αRes cells. Control cells, IRE1αKo cells, and IRE1αRes cells were infected with ZIKV at an MOI of 3 and were collected at 24 h p.i. for Western blotting to detect the expression levels of SCD1. (G) Impact of IRE1α inhibitors on SCD1 expression levels. A549 cells were treated with 30 μM GSK163 or 4μ8c and infected with ZIKV (MOI, 3) at 1 h posttreatment. Then, the cells were collected at 24 h p.i. for Western blotting to measure the expression levels of SCD1. (H) SCD1 expression levels in XBP1 knockdown cells. shScr-, shXBP1-1-, and shXBP1-2-expressing cells were infected with ZIKV at an MOI of 3 and harvested at 24 h p.i. for Western blotting to measure the expression levels of SCD1. (I) Knockdown efficiency of SCD1-specific siRNAs. A549 cells were transfected with siSCD1 for 48 h. Then, the cells were collected to measure the expression of SCD1 by Western blotting. (J to L) Effect of SCD1 knockdown on ZIKV replication levels. A549 cells were transfected with siSCD1, followed by ZIKV infection at an MOI of 3. Then, the cells and supernatants were harvested at 24 h p.i. to detect viral RNA levels (J), viral E protein levels (K), and viral yields (L) as described above. Human β-ACTIN mRNA level was measured as an internal control for qRT-PCR. Data are representative of those from three independent experiments. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (two-tailed Student t test).